Inappropriate responses of the immune system may cause stressful symptoms to the involved organism. Exaggerated immune answers to foreign substances or physical states which usually do not have a significant effect on the health of an animal or human may lead to allergies with symptoms ranging from mild reactions, such as skin irritations to life-threatening situations such as an anaphylactic shock or various types of vasculitis. Immune answers to endogenous antigens may cause autoimmune disorders such as systemic lupus erythematosus, idiopathic autoimmune hemolytic anemia, pernicious anemia, type 1 diabetes mellitus, blistering skin diseases and different kinds of arthritis.
Immune responses occur in a coordinated manner, involving several cell types and requiring communication by signaling molecules such as cytokines between the cell types involved. This communication may be influenced or inhibited by, e.g., interception of the signals or block of the respective receptors.
Cytokines are secreted soluble proteins, peptides and glycoproteins acting as humoral regulators at nano- to picomolar concentrations behaving like classical hormones in that they act at a systemic level and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. Cytokines differ from hormones in that they are not produced by specialized cells organized in specialized glands, i.e. there is not a single organ or cellular source for these mediators as they are expressed by virtually all cells involved in innate and adaptive immunity such as epithelial cells, macrophages, dendritic cells (DC), natural killer (NK) cells and especially by T cells, prominent among which are T helper (Th) lymphocytes.
Depending on their respective functions, cytokines may be classified into three functional categories: regulating innate immune responses, regulating adaptive immune responses and stimulating hematopoiesis. Due to their pleiotropic activities within said three categories, e.g., concerning cell activation, proliferation, differentiation, recruitment, or other physiological responses, e.g., secretion of proteins characteristic for inflammation by target cells, disturbances of the cell signaling mediated by aberrantly regulated cytokine production have been found as a cause of many disorders associated with defective immune response, for example, inflammation and cancer.
Interleukin-32 (IL-32, also known as Natural killer cells protein 4) is a recently discovered cytokine with important functions in host defense and innate immunity. The human IL-32 gene is located on chromosome 16p13.3. Besides human and simian, so far bovine, swine and equine homologues have been found, however no mouse homologues are known so far. Six IL-32 isoforms are known, produced by alternative splicing (Chen et al., Vitam Horm 74 (2006), 207-228). The longest isoform, IL-32gamma (IL-32γ or IL-32g), comprises 234aa (UniProtKB/Swiss-Prot identifier: P24001-1). The second isoform, also known as IL-32beta (IL-32β or IL-32b; UniProtKB/Swiss-Prot identifier: P24001-2) has 188aa. The third isoform of 178aa is also known as IL-32delta (IL-32δ or IL-32d; UniProtKB/Swiss-Prot identifier: P24001-3). IL-32alpha (IL-32α or IL-32a) of 131aa is the fourth isoform (UniProtKB/Swiss-Prot identifier: P24001-4). Isoform 5 (UniProtKB/Swiss-Prot identifier: P24001-5) and isoform 6 UniProtKB/Swiss-Prot identifier: P24001-6) have 168aa respective 179aa. However, also further isotypes may exist, e.g., a 112 aa potential new isotype has been reported by Imaeda et al., (Mol Med Rep. 4 (2011), 483-487).
The receptor for IL-32 is unknown so far. However, some data exist indicating that IL-32 may be bound and cleaved at the cell membrane by proteinase 3 implicating this molecule as a possible receptor, wherein the produced fragments may have biological activity and activate macrophage inflammatory protein-2 and IL-8 (Dinarello and Kim, Ann Rheum Dis. 65 Suppl. 3 (2006); iii 61-64). IL-32 is implicated as a major controller of inflammatory pathways with a pronounced synergy with TNFα in form of a self-perpetuating loop where IL-32 promotes TNFα expression and vice versa resulting in the amplification of proinflammatory mediators. It has been reported to induce various cytokines such as TNFA/TNF-alpha, IL-1β, IL-6, IL-8, and macrophage inflammatory protein-2 (MIP-2), to activate typical cytokine signaling pathways of NF-kappa-B and p38 MAPK and it is an IL-18 inducible gene (Kim et al., Immunity 22 (2005), 131-142; Netea et al., Proc Natl Acad Sci USA. 105 (2008), 3515-3520; Netea et al., Proc Natl Acad Sci USA. 102 (2005), 16309-16314; Joosten et al., Proc. Natl. Acad. Sci. USA 103 (2006), 3298-3303). Recently, it was also shown that IL-32 increases IFN-γ production by Peripheral Blood Mononuclear Cells (PBMCs; Nold et al., J Immunol. 181 (2008), 557-565; Netea et al., PLoS Med. 3 (2006), e277).
IL-32 has been reported as being produced mainly by NK cells, T lymphocytes, epithelial cells, and blood monocytes stimulated by IL-2 or IFN-γ (Dahl et al., J Immunol. 148 (1992), 597-603; Kim et al., (2005)). Furthermore, IL-32 has been observed to be overexpressed in rheumatoid arthritis (RA) synovial tissue biopsies, wherein the level of IL-32 expression correlated positively with the severity of inflammation (Alsaleh et al., Arthritis Res Ther. 12 (2010), R135; Cagnard et al., Eur Cytokine Netw. 16 (2005), 289-292.). Besides various forms of arthritis, such as rheumatoid arthritis (RA) or ankylosing spondylitis (Ciccia et al., Rheumatology 51 (2012), 1966-1972), which belongs to the family of spondyloarthropathies, IL-32 was found functionally associated with several other inflammatory bowel disease (IBD), myasthenia gravis (MG), chronic obstructive pulmonary disease (COPD), asthma, Crohn's disease, psoriasis, atopic dermatitis and cancer (Alsaleh et al., (2010); Breenan and Beech, Curr. Opin. Rheumatol., 19 (2007), 296-301; Asquith and McInnes, Curr. Opin. Rheumatol., 19 (2007), 246-251; Dinarello and Kim, Ann Rheum Dis. 65 Suppl 3 (2006); iii 61-64; Fantini et al., Inflamm Bowel Dis. 13 (2007), 1419-1423; Lee et al., Oncology Letters 3 (2012), 490-496). The high rate of atherosclerosis in RA suggested also a possible role of IL-32 in the inflammatory pathways of vascular inflammation and atherosclerosis, which implications have been also verified, e.g., by detection of IL-32 expression, with the expression of IL-32β and IL-32γ mRNA significantly enhanced in human atherosclerotic arterial vessel wall (Kobayashi et al., PLoS One. 5 (2010); e9458; Heinhuis et al., Cytokine. (2013), S1043-4666). IL-32 may also play a role in immune responses to tuberculosis (Kundu and Basu, PLoS Med., 3 (2006), e274; Netea et al., 2006). Also, increased transcription of IL-32 has been observed after infection by bacteria and viruses, such as Mycobacterium tuberculosis (Netea et al., 2006) or Influenza A (Li at al., PLoS One. 3 (2008), e1985) indicating its possible role in host defense.
Accordingly, IL-32 represents a not yet fully understood, however, important new therapeutic target and there is requirement for IL-32 specific binding molecules, which neutralize the function of all IL-32 isotypes, selected sub-ranges thereof or singular IL-32 isotypes, e.g., IL-32γ.
First attempts to provide such molecules have been already met. For example, U.S. Pat. No. 7,641,904 B2 by Kim et al. provides murine IL-32 monoclonal antibodies, wherein one of the antibodies selectively recognizes IL-32α, wherein another antibody binds IL-32α, IL-32ß, and IL-32γ. International application WO 2005/047478 describes the generation of murine antibody fragments specific for IL-32α and IL-32β. However, apparently no antibodies specific for IL-32γ have been provided yet.
Furthermore, due to immunological responses to foreign antibodies, as mouse antibodies in humans (HAMA-response; Schroff et al., Cancer Res. 45 (1985), 879-885; Shawler et al., J. Immunol. 135 (1985), 1530-1535), mostly humanized versions of antibodies are used in present therapeutic approaches (Chan et Carter, Nature Reviews Immunology 10 (2010), 301-316; Nelson et al., Nature Reviews Drug Discovery 9 (2010), 767-774). One approach to gain such antibodies was to transplant the complementarity determining regions (CDR) into a completely human framework, a process known as antibody humanization (Jones et al., Nature 321 (1986), 522-525). This approach is often complicated by the fact that mouse CDR do not easily transfer to a human variable domain framework, resulting in lower affinity of the humanized antibody over their parental murine antibody. Therefore, additional and elaborate mutagenesis experiments are often required, to increase the affinity of the so engineered antibodies. Another approach for achieving humanized antibodies is to immunize mice which have had their innate antibody genes replaced with human antibody genes and to isolate the antibodies produced by these animals. However, this method still requires immunization with an antigen, which is not possible with all antigens because of the toxicity of some of them. Furthermore, this method is limited to the production of transgenic mice of a specific strain.
Another method to generate antibodies is to use libraries of human antibodies, such as phage display, as described, for example, for the generation of IL-13 specific antibodies in international application WO 2005/007699. Here, bacteriophages are engineered to display human scFv/Fab fragments on their surface by inserting a human antibody gene into the phage population. Unfortunately, there is a number of disadvantages of this method as well, including size limitation of the protein sequence for polyvalent display, the requirement of secretion of the proteins, i.e. antibody scFv/Fab fragments, from bacteria, the size limits of the library, limited number of possible antibodies produced and tested, a reduced proportion of antibodies with somatic hypermutations produced by natural immunization and that all phage-encoded proteins are fusion proteins, which may limit the activity or accessibility for the binding of some proteins. A further severe drawback of this technique is that the antibodies so produced bear the risk of undesired cross-reactivity against self-antigens and lack the characteristics of evolutionary optimized natural human antibodies produced by the human immune system. Furthermore, such antibodies may not be specific enough because of cross-reactivity with other proteins and/or with the target protein in context with normal physiological environment and function. Similarly, European patent application EP 0 616 640 A1 describes the production of auto-antibodies from antibody segment repertoires displayed on phage. Phage libraries are generated from unimmunized humans in this respect (see, e.g., Example 1; page 16, lines 43-51; Example 2, at page 17, paragraph [0158], lines 57-58). However, also the methods described in this patent application suffer from above mentioned general disadvantages of antibodies generated from phage libraries, in comparison to antibodies produced and matured in a mammalian, i.e. human body.
In view of the above, there is still a need for additional and new compounds for treatment and diagnosis of disorders or conditions associated with detrimental IL-32 activity, like binding molecules of high specificity for IL-32 or specific for a selected range of or a single IL-32 isotype, in particular of antibodies specific for IL-32γ, which are tolerable in humans either for monotherapy or combinatorial approaches.
The solution to this problem is provided by the embodiments of the present invention as characterized in the claims and disclosed in the description and illustrated in the Examples and Figures further below.